Anti phospho β catenin ser552

Soluble CD58 (sCD58), AKT inhibitor (LY294002), and Akt activator (SC-79) were acquired from Med Chem Express (MCE, Shanghai, China). Anti-Oct4 (11263-1-AP), anti-Sox2 (66411-1-Ig), anti-CD24 (18330-1-AP), anti-vimentin (10366-1-AP), and anti-c-Myc (10828-1-AP) were purchased from Proteintech (Wu Han, China). Anti-EPCAM (#2626S), anti-E-cadherin (#3195S), anti-AKT (#9272S), anti-GSK-3β (#5676S), anti-Phospho-GSK-3β (Ser9) (5558S), anti-β-Catenin (#8480S), anti-non-phosphorylated (active) β-catenin (S33/37/T41) (#8814S), anti-phospho-β-Catenin (Ser552) (#9566S), and anti-cyclin D1 (#55506S) were acquired from Cell Signaling Technology. Anti-CD58 (A0806) and anti-phospho-AKT (Ser473) (AP0140) were purchases from ABclonal (Wu Han, China).

Total protein was extracted from NE-4C cells using the NucleoSpin RNA/Protein Kit (Macherey-Nagel). Samples were separated by polyacrylamide gel electrophoresis (PAGE). Primary antibodies used for EP expression levels include rabbit polyclonal anti-EP1, −EP2, −EP3, −EP4 (1:200; Santa Cruz Biotechnology). Detection of rabbit monoclonal anti-Phospho-Histone H3 (Ser10) (1:1000; Cell Signaling) was used as a measure of cell splitting behaviour. Primary antibodies used for β-catenin expression levels were rabbit monoclonal anti-non-phospho (Active) β-catenin (Ser33/37/Thr41) and rabbit polyclonal anti-phospho-β-catenin (Ser552) (1:1000; Cell Signaling). Blots were reprobed with mouse monoclonal anti-β-Actin (1:10,000; Abcam). Visualization of bound anti-rabbit and anti-mouse horseradish peroxidise-conjugated secondary antibodies was achieved by incubation with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detection by Geliance 600 Imaging System (Perkin Elmer).